Immortalized and malignant human prostatic cell lines

ABSTRACT

Immortalized malignant human prostatic epithelial and fibroblast cell lines are described containing DNA of a human papillomavirus (HPV) and similar cell lines containing DNA of a human papillomavirus and an activated viral ras oncogene, such as v-Ki-ras. The cell lines are useful for research on drugs for treatment of prostatic cancer and other diseases. The cell lines are useful for research on causes, treatment and prevention of prostate cancer, benign prostatic hyperplasia, male infertility, birth defects, aging and assessment of environmental toxic agents.

GOVERNMENT RIGHTS

This application was funded under NIH Grant NIH 2 S07RR05656 23. TheU.S. Government has certain rights under this application and any patentissuing thereon.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates to human prostatic epithelial andfibroblast cell lines which have been immortalized. In particular, thepresent invention relates to non-malignant cell lines immortalized withDNA of human papillomavirus (HPV) and malignant cell lines containingDNA of HPV and ras oncogene.

(2) Description of Related Art

Human cells are generally difficult to grow and maintain in long-termcultures in vitro. They have a limited life span in culture, grow for ashort time and usually after 4 or 5 sub-cultures, they undergosenescence and die.

Prostate cancer is the leading cancer in men in the United States, interms of incidence. Thirty-two percent (32%) of all cancers in men arisein the prostate. It is estimated by the American Cancer Society that200,000 new cases of prostate cancer will occur in the U.S. in 1994.Prostate cancer is the second leading cause of death from cancer and38,000 deaths are estimated to occur in 1994. African American men havethe highest incidence of prostate cancer in the world, which is almosttwice as high as that in white men and more than 600 times higher thanin men from Thailand. One in 10 men in the U.S. by age 85 will developprostate cancer in his lifetime. An estimated 11 million men have latentor clinical prostatic carcinoma. Approximately sixty-five (65%) of thecases already have metastatic disease at the time of diagnosis. Thesurvival rate is less than 20% after metastasis.

The causes of prostate cancer are not known at the present time. A studyof the causes, prevention and treatment has been hampered by the factthat no good animal or cell models are available. Although rat prostaticcells have been used extensively for such studies, rat prostate is nothomologous to the human prostate, thus, it is not an ideal system touse.

There is a need for cell lines derived from normal human prostate whichcan be used for studies on the process of prostate cancer development inman and to identify agents which may cause or prevent prostate cancer.

Attempts have been made to immortalize human adult prostatic epithelialcells using a monkey virus (Simian virus SV40; Cussenot, O., et al.,Journal of Urology 143, 881-886 (1991); Kaighn, M. E., et al, CancerResearch, 49, 3050-3056 (1989); Lee et al., Internat. J. Oncol.4:821-830 (1994)). Only a monkey virus (SV40) fetal and adult humanprostatic epithelial cells have been immortalized.

Human papillomavirus-18 (HPV-18) has been shown to immortalize humanepithelial cells such as keratinocytes and cervical cells (Kaur, P., etal., Journal of Virology 62, 1917-1924 (1988); and Woodworth, C. D., etal., Cancer Research 48, 4620-4628 (1988)). The human papillomaviruseshad never been used with human prostatic cells. An abstract waspublished in March 1993 describing an HPV immortalized prostaticepithelial cell line by the inventors and others; however, there was nodisclosed method for producing the cells, particularly the use of avirus carrying a v-ki-ras oncogene for this purpose (Proceedings of theAmerican Association for Cancer Research 34:117 (March 1993)).

There is increasing evidence for the involvement of the ras oncogenesand HPV infection in human prostatic carcinogenesis. Expression ofactivated ras oncogene has been demonstrated in human prostateadenocarcinomas (Viola et al., N. England J. Med. 314:133-137 (1986)).Additionally, a relatively high frequency (27%) of Ki-ras codon 12mutation in human prostate carcinomas has also been detected by PCRamplification (Carter, B. S., et al., Cancer Research 50, 6830-6832(1990)). In separate studies, a high prevalence of high-risk HPV DNA inhuman prostate carcinomas has been described (McNicol, J. P., et al.,Journal of Urology 145, 850-853 (1991)). PCR analysis of type-specificHPV-sequences has been used to assess the prevalence of HPV DNA inprostate tissues from 83 Canadian patients. HPV DNA was found in 35 of56 benign prostatic hyperplasia and in 14 of 27 prostate carcinomas. Atleast in one study, development of disease correlated with both thepresence of HPV sequences and the activation of cellular ras genes(Anwar, K., et al., Cancer Research 52, 5991-5996 (1992)). Anwar et al.examined the frequency of ras mutations and the presence of high-riskHPV DNA sequences in 75 specimens from Japanese patients with prostatecarcinomas using PCR amplification (Anwar, K., et al., Cancer Research52, 5991-5996 (1992)). Mutant ras genes were present in 41% of cases andthe frequency of ras mutations and HPV infection increased in patientswith advanced tumors.

OBJECTS

It is therefore an object of the present invention to provide animmortalized non-malignant human prostatic epithelial cell line.Further, it is an object of the present invention to provide a malignanthuman epithelial cell line derived from the immortalized, non-malignantcell line. Further still, it is an object of the present invention toprovide cell lines which are useful for research on prostatic cancer invitro and for other purposes as set forth in detail hereinafter. Afurther objective of the present invention is a method for conversion ofa non-tumorigenic, immortalized cell into a tumorigenic cell using avirus containing an oncogene, by exposure to radiation or by exposure toa chemical carcinogen. It is also an object of the present invention toprovide methods and kits for screening carcinogenic agents or potentialchemotherapeutic, anti-invasive, anti-metastatic and chemopreventiveagents using an immortalized non-malignant and/or malignant adult humanprostate epithelial cell line.

These and other objects will become increasingly apparent by referenceto the following description and the drawings.

DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B are photomicrographs showing a comparison of themorphology of HPV-18 DNA transfected adult human prostatic epithelialclonal line C-1 (FIG. 1A) with Ki-MuSV-transformed HPV-18 DNAtransfected C-1 clonal line (FIG. 1B). FIG. 1C is a photomicrographshowing hematoxylin-and-eosin stained section of a poorly-differentiatedcarcinoma produced by inoculation of the Ki-MuSV-transformed HPV-18 DNAtransfected C-1 clonal line into adult 129J nude mice (x 120).

FIG. 2 shows a Southern blot showing a hybridization analysis ofBamHI-digested genomic DNA from HPV-18 transfected human prostateepithelial (HPE) cell clones using a 1.5-xb XbaI DNA probe that spansthe 3' end of E6, the entire E7, and the 5' end of E1 coding regions ofHPV-18 as shown in FIG. 2A. The arrowhead shows an approximately 4.2-kbfragment that is indicative of the presence of the HPV genome in theappropriate clones. The locations of the molecular weight markers(M_(r)) used are shown on the left. Lane 1: HPV-18 transfected HPE cloneA-2. Lane 2: HPV-18 transfected HPE clone C-I. Lane 3: Early passage HPEcells (negative control). FIG. 2A is a plasmid diagram of plasmidpSHPV-18m which contains a single copy of the HPV genome inserted intothe Eco R1 site of pSV2neo.

FIGS. 3A and 3B are Western immunoblot showing an analysis of HPV-18 E7protein and cellular p53 protein in the HPV-18 transfected humanprostate epithelial (HPE) clones. The appropriate cell extracts wereanalyzed using rabbit antisera against either the HPV-18 E7 protein(FIG. 3A) or the cellular p53 protein (FIG. 3B). The molecular weightmarkers (M_(r)) used were 97.4, 69, 46, 30 and 14.3 kilodaltons. Thearrowheads show the approximately 15-kD E7 protein (FIG. 3A) and the53-kD p53 protein (FIG. 3B). Lanes 1: HPV-18 transfected HPE clone A-2.Lanes 2: HPV-18 transfected HPE clone C-1. Lanes 3: SV40 ori-transfectedHPE clone. Lanes 4: HeLa cells.

FIG. 4 is a Western immunoblot showing expression of p21 ras protein inKi-MuSV transformants. Subconfluent cultures of a Ki-MuSV transformedhuman keratinocyte line (lane 1), HPV-18 transfected HPE clone C-1 line(lane 2), Ki-MuSV infected HPV-18 HPE clone C-1 line (lane 3) and tumorline 129 Nu 3120-3 (lane 4) were rinsed with phosphate-buffered salineand lysed with a buffer containing 50 mM Tris-HCl, pH 7.5, 150 mM NaCl,0.1% SDS, 1.0% Triton X-100, and 1.0% Na deoxycholate. The proteinconcentration of the lysates was determined and equivalent amounts ofprotein from each cell line were immunoprecipitated with either apan-reactive mouse antibody against p21. The resultingimmunoprecipitates were fractionated on a 12.5% polyacrylamide-SDS gel.After electrophoresis, proteins on the gel were electrotransferred ontoa nitrocellulose membrane which was then reacted against the sameantibody against p21. The immunocomplex on the nitrocellulose membranewas visualized by a subsequent reaction with ¹²⁵ I-labeled protein A.Positions of the endogenous wildtype p21 doublet (arrowhead) and theexogenous activated p21 doublet (arrow) are indicated. The molecularweight (M_(r)) markers used were 97.4, 69, 46, 30 and 14.3 kilodaltons.

DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention relates to an immortalized non-malignant ormalignant human prostatic epithelial or fibroblast cell line free ofother cell lines and containing DNA of a human papillomavirus.

The primary prostatic cells to be immortalized can be from variousdonors and cell sources. The cells can be epithelial cells andfibroblasts. The epithelial cells include stem, basal, intermediate anddifferentiated luminal cells.

Alternatively, the non-malignant immortalized cells of the presentinvention can be made malignant by exposure to a virus, to radiation orto chemical carcinogens including for exampleN-methyl-N'-nitro-N-nitrosoguanidine (MNNG) or N-methyl-N-nitrosourea(NMU) and by promotion with tumor promoters such as12-O-tetradecanoylephorbol-13-acetate (TPA). All of this is well knownto those skilled in the art.

Further the present invention relates to a method for producing animmortalized malignant human prostatic epithelial or fibroblast cellline which comprises: providing non-malignant human prostatic epithelialor fibroblast cells with DNA of a human papillomavirus virus; andtransforming the human prostatic epithelial or fibroblast cells with DNAof a virus which causes the non-malignant cell line to become malignantto thereby produce the malignant cell line.

Further still, the present invention relates to a method for producingan immortalized human prostatic epithelial or fibroblast cell line whichcomprises: providing non-malignant human prostatic epithelial cells in aculture; and transfecting the epithelial cells in the culture with DNAof a human papillomavirus so that the cell line is immortalized. Thiscell line is used to produce the malignant cell line.

Human prostatic epithelial or fibroblast cells are immortalized usingthe entire Human Papillomavirus (HPV) genome or portions thereof. TheHPV DNA may be obtained from different strains of HPV which areassociated with cancer especially cancer of the breast, cervix, prostateor urogenital tract. The HPV DNA may be obtained from different strainsof HPV which are isolated from malignant or benign tumors taken from thebreast, cervix, prostate or urogenital tract of humans. Examples of suchstrains of HPV include but are not limited to HPV-18, HPV-31, HPV-33 andthe like.

In one embodiment the cells are immortalized using the entire HPV genomefrom HPV-18, or HPV-31 and the like. In another embodiment, the humanprostatic epithelial or fibroblast cells are immortalized using at leastthe E7 DNA portion of the genome or at least the E6 DNA portion of thegenome or combinations thereof. In one embodiment a DNA sequencehomologous or significantly homologous to the DNA sequence of E6 or E7of HPV is used to immortalize human prostatic epithelial or fibroblastcells. In another embodiment, the cells are immortalized using at leastthe E7 DNA portion in combination with the E6 DNA portion of the HPV-18genome.

Further, the present invention relates to a kit for screening acarcinogenic or chemotherapeutic agent comprising an immortalized humanprostate epithelial or fibroblast cell line or derivative thereofcontaining DNA of a human papillomavirus. Particularly included areother ras oncogenes such as Ki-ras oncogene.

The present invention relates to a method for testing carcinogenicity ofan agent comprising culturing the previously described non-malignantcell line previously described with an agent suspected of beingcarcinogenic and determining formation of an abnormal cellular mass bysaid cell line, the formation of the abnormal cellular mass beingindicative of carcinogenicity of said agent.

The present invention also relates to a method for testing anti-invasiveactivity of an agent comprising culturing the malignant cell linepreviously described with a potential anti-invasive agent anddetermining invasive ability of said cell line being indicative of ananti-invasive activity of said agent.

The present invention also relates to a method for testinganti-metastatic activity of an agent comprising growing the malignantcell line previously described in nude mice, with a potentialanti-metastatic agent and determining the metastatic ability of thetumor generated from the said cell line being indicative of ananti-metastatic activity of said agent.

The present invention also relates to a method for testinganti-angiogenic activity of an agent comprising growing the cell linepreviously described in vitro, in nude mice and in chick chorioallantoicmembrane with a potential anti-angiogenic agent and determiningangiogenic ability of said cell line being indicative of ananti-angiogenic activity of said agent.

The preferred cell lines are deposited with the American Type CultureCollection in Rockville, Md. The immortalized cell line with HPV-18 isdeposited as ATCC CRL-11609 (RWPE-1). The immortalized, and Ki-rastransformed malignant cell line is deposited as ATCC CRL-11610 (RWPE-2).The deposits are under the Budapest Treaty and were made on Apr. 19,1994. ATCC CRL-11609 and ATCC CRL-11610 were deposited on Apr. 7, 1994with the American Type Culture Collection, 12301 Parklawn Drive,Rockville, Md. 20852.

V-Ki-ras from Ki-MuSV (Kirsten murine sarcoma virus) is a mutated rasviral oncogene (v-ras) with the ability to induce a malignanttransformation of the cell line. The C-ras are normal proto-oncogeneswhich are expressed transiently in normal prostatic epithelial cells. Inthe preferred form, the Ki-MuSV is used as a hybrid with baboonendogenous virus (BaEV).

This invention demonstrates the first malignant transformation of adulthuman prostatic epithelial cells in culture by a combination of viraloncogenes. The invention particularly demonstrates the sequentialinvolvement of HPV-18 and activated Ki-ras in the malignant conversionof human prostate epithelial cells and in particularly offers also aunique in vitro model system for further analysis of molecular eventsunderlying prostate carcinogenesis.

In order to obtain further insights into the mechanism of prostaticcarcinogenesis, newly-developed tissue culture methods were used toascertain whether HPV-18 or Kirsten murine sarcoma virus (Ki-MuSV)containing an activated Ki-ras oncogene could confer a malignantphenotype on early passage human prostate epithelial cells. The presentinvention shows the immortalization of normal human prostatic epithelialcells by polybrene (Hexadimethrine bromide from Aldrich Chemical Co.,Product No. 10768-9, Milwaukee, Wis.) induced DNA transfection of theHPV-18 genome and subsequent conversion of such non-tumorigenic butimmortalized cells into tumorigenic cells by the introduction of anactivated Ki-ras oncogene.

The present invention provides direct evidence for the sequentialinvolvement of HPV-18 and Ki-ras in the malignant conversion of humanprostatic epithelial cells and offers a unique in vitro model system forfurther analysis of molecular events underlying prostate carcinogenesis.

EXAMPLE 1

Epithelial cells were derived from the histologically normal prostate ofa 54 year old white male undergoing a cystectomy for bladder carcinoma.The tissue was minced and digested in RPMI-1640 medium (GIBCO 11875-051,Grand Island, N.Y.) containing 5% fetal bovine serum and 400 u/mlcollagenase (Webber, M. M., In Vitro Models for Cancer Research. In M.M. Webber, L. I. Sekeley, Eds., Vol. V. pp. 3-24 (1988)). The isolatedprostatic epithelial acini were plated into fibronectin and type IVcollagen coated plates in keratinocyte serum-free medium containing 25mg bovine pituitary extract and 2.5 μg EGF/500 ml K-SFM (GIBCO k-SFM,10005-018, Grand Island, N.Y.). Secondary cultures grown in keratinocyteserum-free medium were used for immortalization.

Source of HPV type 18 DNA: Plasmid pSHPV-18m contains a single copy ofthe HPV 18 genome inserted into the Eco RI site of pSV2 neo as describedby Woodworth et al, Cancer Res 48:4620-4628 (1988). A genomic map ofpSHPV-18m is shown in FIG. 2A.

Transfection: Polybrene-induced DNA transfection was carried out asdescribed in Rhim et al., Oncogene 4:1403-1409 (1989). Cells weretransfected with 10 μg of DNA using polybrene at a concentration of 10μg/ml and incubated at 37° C. overnight. The cells were then shockedwith 30% DMSO for 4 minutes. Five days after transfection the cells weresubcultured at a 1:2 ratio approximately weekly. The cell culture mediumwas changed twice weekly.

                  TABLE 1                                                         ______________________________________                                                    Passages             Nude mice                                                in          Activated                                                                              with                                                     culture     Ki-ras   tumors/mice                                  Cells       (number)    expression                                                                             inoculated*                                  ______________________________________                                        Parental    <5          ND       ND                                           cells                                                                         plus Ki--MuSV                                                                             <5          ND       ND                                           plus HPV-18 >30         -        0/5                                          plus HPV-18 >30         +        4/5**                                        + Ki--MuSV                                                                    ______________________________________                                         *Nude mice were inoculated with 10' cells.                                    **tumors were reestablished in tissue culture and confirmed to resemble       the cells of origin by karyological analysis.                                 ND = not done.                                                           

Table 1 shows the biological properties of adult human prostaticepithelial cells exposed to Ki-MuSV and/or HPV-18 DNA.

EXAMPLE 2

A number of cell lines were isolated from individual colonies usingcloning cylinders and stored frozen in liquid nitrogen. Two clones withselected, designated A-2 and C-1, for further characterization. Both A-2and C-1 cells had the typical polygonal arrangement of epithelial cells(FIG. 1A) but were less polygonal than the parental cells. Both celllines have apparently an unlimited life-span; each has been successfullysubcultured for more than 30 passages over the course of 1 year with noevidence of decreased proliferative capacity as shown in Table 1.

To confirm that the two clones do contain the transfected HPV-18 DNA,Southern blot hybridization analysis (Tanaka, K., et al., Cell35:457-465 (1983)) was carried out using genomic DNA that had beendigested with BamHI and a 1.5-kb XbaI DNA probe that spans the 3' end ofE6, the entire E7, and the 5' end of E1 coding regions as shown in FIG.2A. The detection of an approximately 4.2-kb fragment in both clones A-2and C-1, but not in the early passage parental cells demonstrates thatHPV-18 sequences have been integrated into both cell clones as shown inFIG. 2. FIGS. 1A to 1C show the morphology of the cell lines.

As evidence for the acquisition of the morphological change being theconsequence of expression of HPV-18 functions, the presence of theHPV-18 E7 gene product was determined by Western immunoblot analysis thepresence of the HPV-18 E7 gene product using a rabbit antibody (Maloy,W. L., et al., Proceedings of the National Academy of Science USA81:1216-1220 (1984)). Identification of an approximately 15-kD componentin both clones A-2 and C-1, that is also present in HeLa cells but notin an SV40 ori-transfected human prostatic epithelial clone, isconsistent with transformation requiring at least the expression of theE7 protein. This is seen in FIG. 3A. Interestingly, while the tumorsuppressor protein p53 is detected in abundance in the SV40ori-transfected clone, presumably because of its stabilization bybinding to the SV40 T-antigen, it is not detectable in either of the twoHPV-18 transfected clones. This is seen in FIG. 3B. This latterobservation is consistent with E7 destabilizing p53, a process which hasbeen proposed to result in the loss of tumor suppressor activity.

Evidence for the human origin of the two lines was obtained by isoenzymeanalysis and cell membrane species-specific immunofluorescence.Moreover, both cell lines showed human karyotype with a Y chromosome.When clone C-1 was analyzed at passage 11 and clone A-2 at passage 15,both were aneuploid and had five and nine marker chromosomes,respectively.

Immunocytochemical analysis showed that both cell lines expressedcytokeratins 8 and 18, which are specific epithelial cell markers.Prostate-specific antigen was strongly present in parental cells, inearly passages of both clones as well as in later passages of cell linestreated with the androgen 5α-dihydrotestosterone.

When analyzed for biological properties, it was observed that neithercell line grew in soft agar or produced tumors in 129J nude mice evenwhen 10⁷ cells were injected as shown in Table 1. Thus, HPV-18 infectionwas associated with the continued proliferative capacity of adult humanprostatic epithelial cells in culture without the concomitantacquisition of a neoplastic phenotype.

In attempts to alter the growth properties of human prostatic epithelialcells, Ki-MuSV was used, since activated Ki-ras oncogene has beendetected in human prostate carcinomas (Viola et al., N. Engl. J. Med.314:133-137 (1986); Carter, B. S. et al Cancer Research 50, 6830-6832(1990); and Konishi, N., et al., Cancer 69, 2293-2299 (1992), andHPV-18, which has also been detected in human prostate carcinomas(McNicol, N. J., et al., Journal of Urology 145, 850-853 (1991); andAnwar, K., et al., Cancer Research 52, 5991-5996 (1992).

Ki-MuSV was produced in human nonproducer cells by superinfection withthe baboon endogenous virus (BaEV) (Rhim, J. S., et al., Science227:1250-1252 (1985). The cells are neomycin resistant. At passage 12,the HPV-18 immortalized prostatic cell line was transformed by infectionwith Ki-MuSV. The characteristics of the resulting immortalizedHPV-18+Ki-MuSV cell line were determined and compared to parental cells,the HPV-18 immortalized cell line or to Ki-MuSV infected cells. Neithercontrol parental nor Ki-MuSV infected cells could be propagated seriallybeyond five subcultures.

In contrast, infection of the HPV-18 transfected C-1 line at passage 12with Ki-MuSV, pseudotyped with baboon endogenous virus (Table 1) tofacilitate entry into human cells, resulted in a marked alteration incell morphology. Two to three weeks after infection, the cells began topile up in focal areas as shown in FIG. 1B. The absence of anydetectable alteration induced by the helper virus alone implied thatKi-MuSV was responsible for inducing the transformed morphology.

The presence and expression of the Ki-ras oncogene in the transformantwas confirmed by Western immunoblot analysis using a pan reactive mouseantibody against the ras p21 protein. Using this analysis, the C-1 clonewas found to express a low level of the endogenous p21 protein asindicated on a SDS-polyacrylamide gel by a characteristic doublet (FIG.4, lane 2) which represents the phosphorylated and non-phosphorylatedforms of the gene product (Rhim, J. S., et al., Science, 232:385-388(1986)). In addition to the endogenous p21, the Ki-MuSV transformed C-1line also expressed a high level of the activated Ki-ras gene productwhich is distinguishable by its slower migration on SDS-polyacrylamidegels (FIG. 4, lane 3). The expression of the activated ras gene in thetransformant is further supported by the presence of the transfectedv-Ki-ras gene, as shown by Southern blot hybridization analysis (datanot shown). HPV-18 DNA analysis indicated that its copy number in theKi-MuSV transformed C-1 cells has remained unchanged (data not shown).

EXAMPLE 3

When adult 129J nude mice were inoculated subcutaneously with 10⁷Ki-MuSV-transformed C-1 cells, tumors developed within 3-weeks at thesite of inoculation as shown in Table 1. Microscopic examination ofsections of these tumors revealed poorly differentiated carcinomasconsistent with neoplastic transformation of the human prostateepithelial cells as shown in FIG. 1C. The tumor consists of large,poorly-differentiated epithelial cells with hyperchromatic,irregularly-shaped nuclei. The presence of a high mitotic index isconsistent with rapid cell division. Structures resembling epithelialpearls are evident suggesting squamous cell differentiation. Smallnondescript cells displaying little cytoplasm are admixed amongst thetumor cells.

Tumors were re-established in culture and confirmed as human epithelialcells by isoenzyme and cytokeratin analyses. Their identity with thecells of origin was determined by karyological analysis. The culturedtumor cells continued to express the activated Ki-ras gene product, asconfirmed by Western immunoblot analysis (FIG. 4, lane 4). Prostatetumor cell lines were then reinjected into athymic nude mice, resultingin rapid tumor formation at a frequency of 100% (5/5). In contrast,subcutaneous inoculation of 10⁷ parental C-1 cells produced no tumorsduring the observation period of six months.

The present invention represents the first malignant transformation ofadult human prostate epithelial cells in culture and supports previousepidemiological studies that have implicated the Ki-ras oncogene and HPVinfection in prostatic carcinogenesis. It has been demonstrated thatexpression of the v-Ki-ras gene in an HPV-18 immortalized adult humanprostatic epithelial cell line facilitates malignant transformation. Twoor more alterations in cellular growth properties seem to be required.The process was indicated by the acquisition of an unlimited growthpotential as a result of HPV-18 gene expression. In addition toacquiring a continuous proliferation capacity, the HPV-18 transfectedhuman prostatic epithelial cells showed an abnormal karyotype at theearliest measurable time. Nonetheless, they lacked the ability to induceprogressively growing tumors in nude mice. Thus, the alterations inducedby HPV-18 DNA transfection are necessary but by themselves areinsufficient to induce progression to the neoplastic state. Variousvirus can induce a cell to become tumorigenic, e.g. oncogenicretrovirus.

Superinfection of early passage HPV-18 immortalized adult human prostateepithelial cells with Ki-MuSV, a virus with an activated Ki-ras oncogene(v-Ki-ras), resulted in further changes in their growth properties.Expression of the activated ras-21 protein, morphological alteration,increase in genetic instability and ability to induce squamous cellcarcinomas in athymic nude mice appeared to be concomitantly-acquiredproperties associated with the addition of the v-Ki-ras oncogene. Thesignificance of the combined action of HPV-18 and the v-Ki-ras oncogenein the induction of malignant transformation of human prostaticepithelial cells is emphasized by the finding that the v-Ki-ras genealone cannot induce neoplastic transformation unless the cells have beenimmortalized by HPV-18. Thus, the v-Ki-ras oncogene has the ability tocomplement with HPV to induce full transformation.

The results show that successive changes are required to inducemalignant transformation of human prostatic epithelial cells of adultorigin, the natural target of prostate carcinogenesis. The multi-step invitro model is invaluable for defining the molecular events underlyingthe development of human prostate cancer.

The following points demonstrate the significance of the novel malignantand non-malignant cell lines:

1. Specimens of normal human prostate are very difficult to obtainbecause of the availability and consent concerns.

2. The non-malignant cell lines were immortalized by using the humanpapillomavirus (HPV) DNA. The importance of this rests in the fact thatcertain HPV's (HPV-16, HPV-18) are sexually transmitted and areassociated with the development of cancer of the cervix. About 95% ofwomen with cervical cancer are found to be positive of HPV. It thereforestands to reason that in some genital cancers in men, these sexuallytransmitted HPV's may also be involved. It was for this reason that HPVDNA was used.

3. HPV-18 DNA has been detected in samples of human prostate cancers.

4. No other HPV-immortalized, human prostatic cell lines have beendeveloped.

5. Assuming that HPV's are involved in human prostate cancer, then thecell models will also be extremely useful for studying the interactionsbetween HPV and prostatic cells during the cancer development process.

6. Both types of cell lines (malignant and non-malignant) are useful.The non-malignant cell lines do not form tumors and retain manyproperties of normal cells. Therefore, they can be used in place ofnormal cells for research.

7. The use of human cell models is much more appropriate than using rator mouse cells, data from which then have to be extrapolated to thehuman condition.

8. The use of cell models for research is preferred as much as possiblein order to avoid unnecessary use of whole animals.

The applications for the cell lines are in the areas of cancer research;research studying aging; cancer prevention; role of diet and nutritionin cancer; cell physiology: studies on the mechanisms of secretion; cellbiology; biochemistry; studies on cell differentiation; developmentalbiology; and all biomedical sciences.

Thus the cell lines can be used in:

1. Cancer Prevention: as models for the discovery and testing of agentswhich may have the ability to prevent prostate cancer and benignprostate hyperplasia (BPH).

2. Cancer Detection and Diagnosis: as models for discovering new markersfor early detection of prostate cancer and BPH.

3. Basic Research: The cell models can be used for a large variety ofstudies, for example:

(a) identification of agents which cause prostate cancer and BPH.

(b) the origin and the process of cancer development in the humanprostate.

(c) mechanisms of tumor progression and its control.

(d) multistep process of carcinogenesis, especially as we have thenormal, immortalized and malignant cells derived from the same prostate.

(e) for the identification of specific genetic defects which lead to thedevelopment of prostate cancer.

(f) for studies on why prostate cancer does not respond well tochemotherapy.

(g) mechanisms of drug resistance in prostate cancer and how tocircumvent resistance.

(h) basic understanding of prostate physiology and its role inreproduction.

4. Models for Risk Assessment of Reproductive Toxicity:

Reproductive toxicity may result in infertility in men, spontaneousabortions in their female partners and birth defects in their offspring.The cells can be used as (a) a model for identification of reproductivetoxins which may be secreted into the seminal fluid and thus damagesperm, resulting in birth defects.

(b) a model for detection of exposure to environmental and industrialreproductive toxins.

(c) a model for detection of reproductive toxicity in men exposed tochemicals in chemical warfare.

(d) a model for infertility and for birth defects in the offspring ofmen exposed to toxic chemicals in the industry and in chemical warfare.

5. Models for Alternatives to Animal Testing:

There is presently a great deal of concern about the use of animals fortesting products for human use. With the upsurge of interest in theAnimal Rights Movement, considerable emphasis is being placed on thedevelopment of cell culture models as alternative to using animals fortesting. The drug and the cosmetic industry and manufacturers ofhousehold and industrial chemicals are interested in using cell culturemodels, especially those derived from human cells, to test for humantoxicity of their products.

6. Cancer Treatment: (a) as cell systems for the discovery of new drugssuch as cisplatin and taxol, for the treatment of prostate cancer andBPH.

(b) as cell models for examining the efficacy of single drugs andcombination of drugs and/or radiation for the treatment of prostatetumors.

(c) as cell systems for identification of anti-invasive agents such asprotease inhibitors and other agents which can specifically preventinvasion and thus, can be used to prevent cancer from invading theneighboring tissues.

(d) as cell models for identification of anti-metastasis agents, whichcan specifically prevent cancer from metastasizing and spreading todifferent organs.

7. Identification of Potential Chemotherapeutic Drugs: These cells areuseful for screening chemicals suitable for the treatment of cancer andrelated diseases, by growing them in vitro in medium containing thechemical to be tested and then, after a suitable period of exposure,determining whether and to what extent cytotoxicity has occurred, e.g.by trypan blue exclusion assay or related assays (Paterson, MethodsEnzymol. 58:141-152 (1979)), or by growth assays such as colony formingefficiency (MacDonald et al., Exp. Cell. Res. 50:471-473 (1968)), all ofwhich are standard techniques well known in the art.

Similarly, potential chemotherapeutic drugs may be screened in vivo insuitable animal models. The tumorigenic human prostate epithelial celllines are injected subcutaneously into athymic nude mice. Potentialchemotherapeutic drugs are injected at various time intervals and doses.Animals are examined weekly for the presence of tumors for a period offour or more months.

8. Studies of Metabolism of Carcinogens and Other Xenobiotics:Carcinogens and other xenobiotics may be added to the growth medium ofcultures of these cells and then the appearance of metabolic products ofthese compounds may be monitored by techniques such as thin layerchromatography or high performance liquid chromatography and the like.The interactions of the compounds and/or their metabolites with DNA canthen be examined.

9. Studies of DNA Mutagenesis: Substances known or suspected to bemutagens may be added to the growth medium of cell cultures, especiallythe immortalized RWPE-1 cells, and then mutations may be assayed, e.g.,by detection of the appearance of drug resistant mutant cell colonies(Thompson, Methods Enzymol., 48:308-322 (1979)). Similarly,cell-mediated DNA mutagenesis, by cocultivating the cells with celltypes known or suspected to be capable of activating mutagenic compounds(Hsu et al., Proc. Natl. Acad. Sci. USA 75:2003-2007 (1978)).

10. Studies of Chromosome Damaging Agents: Substances known or suspectedto cause chromosomal damage may be added to the culture medium of thesecell lines, and then the extent of chromosomal damage may be measured bytechniques such as measurement of the frequency of sister chromatidexchange (Latt et al. In: S. Wolff (ed.), Sister Chromatid Exchanges,New York: Plenum Press, pp. 17-40, (1982)).

11. Studies of malignant transformation by chemical, physical and viralagents, and transferred genes including other oncogenes in addition to,or in place of the Ki-ras oncogene and high molecular weight genomic DNAfrom tumors, using standard assays such as tumor formation in athymicnude mice.

12. Studies of Cellular Responses to Growth Factors, Cytokines, TumorPromotors/Enhancers, or Hormones and Production of Growth factors,Cytokines, Tumor Promotors/Enhancers, or hormones: Identification andpurification of novel growth factors, cytokines, tumorpromotors/enhancers, or hormones important for growth anddifferentiation of human prostate epithelial cells. These cells areparticularly useful for such an application since they grow inserum-free media. Therefore, responses to growth factors, cytokines,tumor promotors/enhancers, or hormones can be studied in preciselydefined growth media and any factors produced by the cells may beidentified and purified without the complication of the presence ofserum. In addition to the isolation and characterization of growthfactors, cytokines, tumor promotors/enhancers or hormones producedendogenously by the cells of the present invention, exogenous sources ofgrowth factors, cytokines, tumor promotor/enhancers or hormones areadded and the effect on growth and differential determined. Moreover,various chemical agents are added in the presence of the endogenous orexogenous growth factors, cytokines, tumor promotors/enhancers orhormones and the effect on cell growth and differential determined. Thechemical agents may be a chemotherapeutic agent, a carcinogenic agent orany chemical agent known or suspected of having an effect on cell growthand/or cell differentiation.

13. Use of recombinant DNA expression vectors to produce proteins ofinterest. For example, the gene encoding a protein of therapeutic valuemay be recombined with controlling DNA segments (i.e. containing apromoter with or without an enhancer sequence), transferred into thecell (e.g. by polybrene transfection) and then the protein produced maybe harvested from the culture supernatant or a cellular extract byroutine procedures well known in the art.

14. Characterization of Cell Surface Antigens or Receptors: The cellsare incubated with an antibody against the cell surface antigen orreceptors of interest, and then reacted with a second antibody which isconjugated to a fluorescent dye. The cells are then evaluated using afluorescence activated cell sorter to determine whether they arefluorescent and therefore possess the cell surface antigen or receptors.Novel surface antigens or receptors are purified and isolated from thecells of the present invention by immunoaffinity chromatography usingantibody specific for the antigen or receptor, using the specific ligandto the receptor or using other chromatographic techniques known in theart.

15. Cell-cell hybrid studies for identification of tumor suppressoractivity (Stanbridge et al., Science 215:252-259 (1982)). To determinewhether immortalized non-malignant cell lines contain tumor suppressorgenes, they are fused to malignant tumor cells. The presence of tumorsuppressor genes in hybrid cells is indicated by loss of malignantproperties, i.e., loss of in vitro characteristics of cancer cells, suchas loss of density dependent inhibition of growth, growth in agar,stimulation of growth by autocrine secretion of growth factors and lossof ability to form tumors in athymic nude mice and chick chorioallantoicmembrane.

16. Identification of novel genes, including transforming genes innaturally occurring cancers, growth factor genes, oncogenes, tumorsuppressor genes, using standard molecular biological techniques (Daviset al., Methods in Molecular Biology, New York: Elsevier, 167-189(1986)) and techniques such as cDNA substraction cloning and the like.

17. Studies of cellular Biochemistry, including changes in cellularphysiology such as changes in intracellular pH and calcium levels andthe like, as correlated with cell growth and action of exogenous agents.To study intracellular pH and calcium levels, cells in suitable culturevessels are exposed to fluorescence emissions are detected with afluorescence spectrophotometer (Grynkiewicz et al, J. Biol. Chem.260:3440-3450 (1985)).

18. Studies of intercellular communication e.g., by dye scrape loadingassays. To determine whether the cells growing in vitro have the abilityto communicate via gap junctions, the cultures may be scraped, e.g.,with a scalpel, in the presence of a fluorescent dye in the growthmedium. Cells at the edge of the wound are mechanically disrupted andtherefore take up dye. Intercellular communication is ascertained bydetermining whether cells distant from the wound also contain dye.

The kit for screening carcinogenic or antineoplastic agents and for anyother usage as described herein supra, is easily assembled, comprisingthe cell line(s) of the present invention. Other components routinelyfound in such kits may also be included with instructions for performingthe test.

The fibroblast cells are associated with prostatic epithelial cells andthus the interactions of the cells can be studied using the cells of thepresent invention.

An application by some of the inventors herein is co-pending describingprostatic cells immortalized with Adenovirus 12 and Simian virus 40.

It is intended that the foregoing description be only illustrative ofthe present invention and that the present invention be limited only bythe hereinafter appended claims.

We claim:
 1. An immortalized adult normal non-malignant human prostatic epithelial cell line, free of other cell types and containing DNA of a human papillomavirus, deposited as ATCC CRL-11609.
 2. An immortalized malignant human adult prostatic epithelial cell line, free of other cell types and containing DNA of a human papillomavirus and further containing an activated Ki-ras oncogene or DNA of a virus containing an activated ras (V-ras) which causes the cell line to become malignant, wherein the malignant cell line is deposited as ATCC CRL-11610.
 3. The cell line of claim 2 wherein the v-ras virus is a sarcoma virus.
 4. The cell line of claim 3 wherein the sarcoma virus is a Kirsten murine sarcoma virus.
 5. The cell line of claim 4 wherein the Kirsten murine sarcoma virus is as a hybrid with a baboon endogenous virus.
 6. The cell line of any one of claims 1, 2, 3, 4 or 5 wherein the human papillomavirus is HPV-8.
 7. A method for producing an immortalized, malignant human adult prostatic epithelial cell line which comprises:(a) introducing adult, non-malignant human prostatic epithelial cells which have been transfected with DNA of a human papillomavirus into a culture medium; and (b) and further transforming the adult non-malignant human prostatic epithelial cells with DNA of a ras oncogene or genome of an activated ras containing virus which causes the adult, non-malignant human cell line to become malignant to produce the malignant cell line deposited as ATCC-CRL-11610.
 8. The method of claim 7 wherein the transforming DNA is a genome of a ras-containing retrovirus.
 9. The method of claim 7 wherein the transforming DNA is of a v-Ki-ras containing virus which is a sarcoma virus.
 10. The method of claim 9 wherein the sarcoma virus is a Kirsten murine sarcoma virus.
 11. The method of claim 10 wherein the murine sarcoma virus is a baboon endogenous virus pseudotype.
 12. The method of any one of claims , 7, 8, 9, 10 or 11 wherein the human papillomavirus is HPV-18.
 13. A method for producing an immortalized human adult, prostatic epithelial cell line which comprises:(a) introducing non-malignant human adult, prostatic epithelial cells into a culture medium; and (b) transfecting the epithelial cells with DNA of a human papillomavirus so that the cell line is immortalized, wherein the cell line is deposited as ATCC CRL-11609.
 14. The method of claim 13 wherein the human papillomavirus is HPV-18.
 15. A kit for screening a carcinogenic agent comprising an immortalized human adult, prostate epithelial cell line containing DNA of a human papillomavirus deposited as ATCC CRL-11609 with instructions for use.
 16. A kit according to claim 15 wherein the immortalized cell line is immortalized using HPV-18 DNA as the human papillomavirus.
 17. A kit according to claim 15 wherein the immortalized cell line is non-malignant.
 18. A kit for screening a chemotherapeutic agent comprising an immortalized human adult, prostate epithelial cell line containing DNA of a human papillomavirus and further containing Ki-ras oncogene or DNA of a virus containing an activated ras (v-ras) which causes the cell line to become malignant and wherein the cell line is deposited as ATCC CRL-11610, with instructions for use.
 19. A method for testing carcinogenicity of an agent comprising culturing a cell line which comprises an immortalized adult, non-malignant human prostatic epithelial cell line free of other cell types and containing DNA of a human papillomavirus and deposited as the cell line ATCC CRL-11609, with an agent suspected of being carcinogenic and determining the formation of malignant cells said formation being indicative of carcinogenicity of said agent.
 20. A method for screening cancer chemotherapeutic and antineoplastic activity of an agent comprising culturing a malignant cell line wherein the cell line contains Ki-ras oncogene or DNA of a virus containing an activated ras which causes the cell line to become malignant and is deposited as ATCC CRL-11610 with said agent and determining growth of said cell line, a lack of growth of said cell line being indicative of an antineoplastic activity of said agent. 